ATP binding cassette (ABC) transporters play critical roles in maintaining sterol balance in higher eukaryotes. The ABCG5/ABCG8 heterodimer (G5G8) mediates excretion of neutral sterols in liver and intestines1–5. Mutations disrupting G5G8 cause sitosterolaemia, a disorder characterized by sterol accumulation and premature atherosclerosis. Here we use crystallization in lipid bilayers to determine the X-ray structure of human G5G8 in a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC sterol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains are coupled to the nucleotide-binding sites by networks of interactions that differ between the active and inactive ATPases, reflecting the catalytic asymmetry of the transporter. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolaemia.

We used sequence and structural comparisons to determine the fold for eukaryotic ornithine decarboxylase, which we found is related to alanine racemase. These enzymes have no detectable sequence identity with any protein of known structure, including three pyridoxal phosphate-utilizing enzymes. Our studies suggest that the N-terminal domain of ornithine decarboxylase folds into a fi/a-barrel. Through the analysis of known barrel structures we developed a topographic model of the pyridoxal phosphate-binding domain of ornithine decarboxylase, which predicts that the Schiff base lysine and a conserved glycine-rich sequence both map to the C-termini of the @strands. Other residues in this domain that are likely to have essential roles in catalysis, substrate, and cofactor binding were also identified, suggesting that this model will be a suitable guide to mutagenic analysis of the enzyme mechanism.

The Janus family of tyrosine kinases (JAKs) 2 are key regulators of cytokine receptor signaling in hematopoiesis and immune responses (1). Of the four mammalian JAK kinases, JAK2 transmits signals for a variety of cytokine receptors, including the erythropoietin receptor (EpoR) that is essential for red blood cell production (2). Upon Epo stimulation, JAK2 activates downstream signaling, such as STAT5, Ras/mitogenactivated protein kinase, and phosphatidylinositol 3-kinase/ AKT pathways (2). Mice deficient in Epo, EpoR, or JAK2 die embryonically due to the absence of definitive erythropoiesis (3-5).In addition to regulation by phosphatases and suppressors of cytokine signaling (6, 7), JAK2 kinase activity is critically controlled by an autoinhibitory mechanism. Like other JAK members, JAK2 contains an N-terminal segment followed by a pseudokinase domain and a C-terminal tyrosine kinase domain. The N-terminal segment, consisting of a FERM (protein 4.1, ezrin, moezin, radixin homologous) domain and an atypical SH2 domain (1), mediates association with the membrane-proximal region of the cytokine receptors (8). Binding of JAK2 through its N-terminal segment to the EpoR is essential for EpoR surface expression (9). The pseudokinase domain is predicted to adopt a kinase fold but lacks residues essential for catalysis (10). Deletion of the pseudokinase domain leads to a marked increase in JAK2 kinase activity and loss of response to cytokine stimulation (11-13). Therefore, this domain is essential for JAK2 autoinhibition and is essential for JAK2 activation upon cytokine stimulation. Consistent with this notion, a point mutation in the JAK2 pseudokinase domain was identified in the majority of myeloproliferative disorder patients, including 90% of polycythemia vera (PV) patients (14 -18). This mutation, V617F, in the presence of a dimerized receptor scaffold, such as the EpoR, resulted in the constitutive activation of JAK2 and downstream signaling effectors (19,20) and caused erythrocytosis in a murine bone marrow transplant model (14,(21)(22)(23). Recently, mutations immediately adjacent to the JAK2 pseudokinase domain in the SH2-pseudokinase domain linker were identified in PV patients and shown to cause constitutive activation of JAK2 and a PV-like phenotype in mice (24 -26). The molecular mechanisms underlying the control of JAK2 activity (i.e. the swift augmentation of its activity upon receptor activation) are poorly understood. The residues involved in the autoinhibition in JAK2 are unknown.In this work, we sought to characterize the regulatory mechanisms controlling JAK2 kinase activity. Using a functional screen for activating JAK2 mutations that signal constitutively, we discovered 13 mutations in the pseudokinase domain and in the SH2-pseudokinase domain linker. These mutations identified specific residues that are important for the inhibition of basal JAK2 kinase activity and for cytokineinduced JAK2 activation. In addition, we showed that the SH2-pseudokinase domain linker is essential for interaction w...

The crystal structure of carboxypeptidase T from Thermoactirzomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the threedimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed p sheet. The active site is located at the C-edge of the central (parallel) part of the B sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.Metallocarboxypeptidases are exopeptidases which contain a zinc ion in the active centre and catalyze the hydrolysis of C-terminal amino acids from polypeptide substrates. Extensive studies of bovine pancreatic carboxypeptidase A (CPA) by different techniques including crystallography, spectroscopy, kinetics and site-directed mutagenesis resulted in the understanding of the general principles of the catalytic mechanism (Christianson and Lipscomb, 1989). Three-dimensional structures have been determined for pancreatic carboxypeptidases A (Rees et al., 1983;Kobe and Goldsmith, 1990) and B (Schmid and Herriott, 1976; Coll et al., 1991) which share a common polypeptide fold but differ in substrate specificity (Hartsuck and Lipscomb, 1971 ;Folk, 1971 polypeptide chain of 326 amino acids and contains one zinc ion (Osterman et al., 1984). The amino acid sequence of CPT, deduced from the gene encoding the protein (Smulevitch et al., 1991), shows only moderate similarity with the CPA and CPB sequences; 30% and 27% identical residues, respectively. CPT exhibits an unusual 'dual'-substrate specificity combining features of both CPA and CPB; it is able to split off hydrophobic and basic amino acids with comparable efficiency (Osterman et al., 1984). As other proteins from Thermoactinomyces, CPT has an increased thermal stability in the presence of calcium ions (Osterman et al., 1984).Structure/functional studies of CPT provide a possibility to study the general principles of catalysis by Zn-carboxypeptidases. On the basis of the comparative primary-structure analysis, all Zn-carboxypeptidases were divided into two groups ; ,,digestive,, and ,,regulatory,, enzymes (Osterman et al., 1992). Pancreatic CPA and CPB appear to be structurally ...

The objective of the Baikal Project is the creation of a kilometer-scale high-energy neutrino observatory: the Gigaton Volume Detector (GVD) in Lake Baikal. Basic elements of the GVD-new optical modules, FADC readout units, and underwater communication systems-were investigated and tested in Lake Baikal with prototype strings in 2008-2010. We describe the results of prototype strings operation and review the preliminary design and expected sensitivity of the GVD telescope.